Fs. Nandel, SAFRANINE-O AS MEMBRANE-POTENTIAL PROBE - A MECHANISTIC STUDY USING FLUORESCENCE SPECTROSCOPY, Indian Journal of Biochemistry & Biophysics, 35(4), 1998, pp. 247-254
Use of safranine-o has been examined as membrane potential probe in 1-
palmitoyl-2-oleoyl-3-phosphatidylcholine (POPC) vesicles both in prese
nce and absence of cholesterol. The fluorescence signal increases in p
resence of vesicles and the increase in fluorescence intensity on hype
rpolarization with valinomycin is diffusion potential dependent. The f
luorescence spectra recorded after time driven experiments reveals the
blue shift in lambda(max) of fluorescence with increasing diffusion p
otential. The fluorescence spectra of vesicles-associated dye is at va
riance with those of the safranine-o in organic solvents. In organic s
olvents with increasing hydrophobic character of the solvent the lambd
a(max) is slightly red shifted. The electronic spectra of the dye mole
cule and the charges on different atomic centers have been calculated
by quantum chemical method GRINDOL. The predicted first excited state
originating from the phenazine moiety is in very good agreement with t
he excitation wavelength. On the basis of charges on various atoms the
binding of safranine with vesicles has been discussed. The nonlinear
behaviour of fluorescence signal with Delta phi, anisotropy measuremen
ts and the computational results, reveal the penetration of bound dye
molecules (along with orientation) as a function of diffusion potentia
l. Addition of microaliquots of 1.5 M K2SO4 to already hyperpolarized
vesicles decreases the fluorescence signal and the fluorescence spectr
a recorded on stabilization of signal after each addition showed a shi
ft in lambda(max) of fluorescence in opposite direction i.e. red shift
ed.